Target cell destruction by alloimmune macrophages may be mediated by a soluble cytotoxic substance that has been designated Specific Macrophage Cytotoxin (SMC). SMC is detected in the supernatant of a mixed culture of alloimmune macrophages and specific target cells. Studies performed with unpurified SMC clearly differentiates it from other reported mouse in vitro cytotoxins. SMC is present in the culture supernatant after only 2 hours of incubation. It is a heat labile, trypsin sensitive cytotoxin which affects only specific target cells. It is a large molecule (MW 150,000 to 200,000), and its cytotoxicity is substantially reduced by adsorption with spcific target cells. SMC is most likely present in vivo during the rejection of Sarcoma I tumor. In order to ascertain this fact for certain a rabbit antipurified SMC is needed. It is planned to obtain purified SMC by submitting concentrated culture supernatants to disc gel electrophoresis followed by the elution of SMC from the gel. Rabbits will be immunized with purified SMC in order to obtain a rabbit anti-purified SMC. Other methods for obtaining a rabbit anti-purified SMC, such as extensive adsorption of rabbit anti-unpurified SMC, will be evaluated. The presence of SMC in vivo will be ascertained using an indirect and direct fluorescent technique employing rabbit anti-purified SMC antibody and tagged goat anti-rabbit gamma globulin. Peritoneal exudate cells will be examined for the presence of SMC during the rejection of Sarcoma I as well as examination of the ascites fluid during the rejection of Sarcoma I by double diffusion in gel using anti-purified SMC. Further, using purified SMC, it is planned to investigate the mechanism of action of SMC on target cells as well as its site of binding. It is anticipated that these studies will substantiate the presence of SMC in vivo during the rejection of tumor and will add to the basic understanding of the mechanism(s) of soluble mediator-induced target cell destruction.